The use of liposomes as carriers of marker molecules for nonisotopic immunoassays is known. See, e.g., U.S. Pat. Nos. 4,704,355; 4,695,554; 4,656,129 and 4,193,983. An important advantage of using liposomes in immunoassays is the ability of liposomes to carry a large number of marker molecules per liposome vesicle, and thereby provide an amplified signal to immunoassays. Immunoassays utilizing liposomes with encapsulated macromolecular markers such as enzymes or small organic marker molecules such as fluorescent or absorbing dyes, spin-labels, metal chelators, and enzyme activators or inhibitors, have been described. See, e.g., Kricka, L. and Carter T., Clinical and Biochemical Luminescence, pp. 153-178 (Marcel Dekker, Inc., New York and Basel, 1982).
Prior to the present invention, chemiluminescent markers such as the acridinium esters described in Ann. Clin. Biochem. 25, p. 27 (1988), Clin Chem. 31, p. 664 (1985), . European Patent Application No. EP 82,636, and U.S. Pat. No. 4,745,181, have only been used as labels for immunoassays by conjugating them directly to biological molecules, such as antigens or antibodies. The lipophilic nature of the prior art acridinium esters and other chemiluminescent compounds render them unsuitable for encapsulation within liposomes because of their rapid leakage through the liposome wall. Additionally, the limited water solubility of prior art acridinium esters and other chemiluminescent compounds only allow the encapsulation of a few marker molecules per liposome vesicle, resulting in relatively low signal amplification.
Accordingly, it is the purpose of the present invention to provide a novel method for detecting an analyte using acridinium esters. It is also a purpose of the present invention to provide novel hydrophilic acridinium esters useful for encapsulation within liposomes for use as chemiluminescent markers.